1. Field of Invention
The present invention relates to a process for production of a desired polypeptide using gene recombination techniques. Preferably the desired polypeptides are physiologically active polypeptides, for examples enzymes such as proteases. For example, the present invention relates a process for production of derivatives of Staphylococcus aureus V8 protease.
As a more specific example, the present invention provides a process for production of an active V8 protease derivative by expressing an insoluble fusion protein of a V8 protease derivative of Staphylococcus aureus origin using an E. coli expressing system, excising the V8 protease derivative from the fusion protein with an ompT protease intrinsic in E. coli in the presence of a denaturating agent and if necessary refolding the V8 protease.
Staphylococcus aureus (S. aureus) V8 protease is one of the proteases secreted into a culture medium by S. aureus V8 strain. This enzyme was isolated and purified by Drapeau, G. R et al. in 1972, as one of the serine proteases, which is secreted into a culture medium of S. aureus V8 strain and specifically cleaves the C-terminal of glutamic acid and aspartic acid (Jean Houmard and Gabriel R. Drapeu (1972), Proc Natl. Acad. Sci. USA, 69, 3506-3509). A DNA nucleotide sequence of the enzyme was determined by Cynthia Carmona et al., in 1987 (Cynthia Carmona and Gregory L. Gray (1987), Nucleic Acids R s. 15, 6757).
It is believed that the present enzyme is expressed as a precursor having 336 amino acid residues, and secreted as a mature protein by deletion of a prepro sequence of 68 amino acid residues from the N-terminal of the precursor. In addition, it is known that the present enzyme has a repeat sequence of proline-aspartic acid-asparagine at the C-terminal region (amino acid numbers 221-256). It is not clear whether or not this repeat sequence is essential for enzymatic activity, and Gray et al. consider that the repeat sequence might function when the enzyme exists as an inactive enzyme prior to secretion.
Although the functions of this enzyme have not been fully analysed, since the enzyme specifically cleaves the C-terminal of glutamic acid and aspartic acid, it is extensively used for determination of an amino acid sequence of proteins. In addition, since the present enzyme acts on a substrate even in the presence of urea (at a concentration of about 2 M), it is used to liberate a desired peptide from its fusion protein, after solubilization, with urea, of a large amount of insoluble fusion protein intracellularly expressed according to a gene recombination technique.
The present inventors successfully used the above-mentioned method to efficiently produce human calcitonin by gene recombination techniques (Japanese Unexamined Patent Publication (Kokai) No. 5-328992, EP528686). In addition, the S. aureus V8 protease was used to excise human glucagon from a fusion protein expressed in the E. coli expression system (Kazumasa Yoshikawa et al. (1992), Journal of Protein Chemistry, 11, 517-525).
As can be seen from the above, the present enzyme has been extensively used for research and production of peptides by gene recombination. However, since the enzyme is purified from a culture medium of S. aureus V8, there are problems in that (1) the enzyme is contaminated with trace amounts of other proteins, (2) the S. aureus V8 is a pathogenic strain, and (3) the product is expensive.